CN1094091A - The seed selection of cordyceps militaris excellent species and high yield cultivating method - Google Patents
The seed selection of cordyceps militaris excellent species and high yield cultivating method Download PDFInfo
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Abstract
The present invention relates to fine-variety breeding and the high yield cultivating method of a kind of Cordyceps militaris (L.) Link..To screen for three times by female kind of original strain process, original seed, the cultivar that conventional monospore or many spores separation method obtain, obtain excellent species.By increasing the rejuvenation of substratum nutritive ingredient, make relatively stable, pure, the difficult degeneration of its bacterial classification kind.This breeding is by appending nutritive medium to substratum, 3-4 stubble entities of can gathering continuously, and its biological transformation ratio can reach more than 150%, has shortened the production cycle more than 20 days, significantly reduces production costs about 40%.
Description
The present invention relates to a kind of edible seed selection and cultivation technique of medicinal fungi held concurrently, a kind of seed selection of cordyceps militaris excellent species and high yield cultivating method.
The patent No. is that the patent of invention of CN88103453.3 discloses a kind of " Cordyceps fungus and cultural method thereof ", from fresh Cordyceps sinensis, separate, through artificial culture, get the parasitic insect of its spore inoculating, produce stroma, form bacterium worm bonded complex body in artificial breeding.Experiment showed, this method not only the cycle long, and sporophore is difficult to form, old friend worker cultivates Cordyceps sinensis and does not obtain success fully.Cordyceps militaris (L.) Link. (cor-dycepsmiLitaris(L) Link) having another name called Cordyceps militaris (L.) Link., is a kind very close with Cordyceps sinensis.This bacterial classification has been deposited in Beijing China Committee for Culture Collection of Microorganisms common micro-organisms center on April 1st, 1993, preserving number CGMCC NO 0193, the inventor once disclosed " method of Cordyceps militaris (L.) Link. bacterium and artificial culture sporophore thereof " in a patent of invention.The cordyceps militaris fruit body that this method obtains by artificial culture, because of it contains distinctive cordycepin, Cordyceps polysaccharide, superoxide dismutase, cordycepic acid etc., so have anticancer, anti-ageing and effect such as nutrition skin, the raw material that can be used for treating multiple disease He do nourishing, protective foods, medicine, beverage, makeup.But bacterial classification of Cordyceps militaris (L.) Link. and cultivating method thereof are desirable not enough at present.For example bacterial classification is impure, degradation phenomena is more serious, it shows as mycelia fine hair weak point, is close to media surface, and the globule appears, the microscopy conidium is many, cylindrical, the mycelia branch is rectangular, behind such strains for cultivation,, be difficult to occur sporophore although the conditions such as suitable temperature, illumination, humidity that give still are difficult for annesl.Influence quality product and output, on cultivation technique, only received a stubble entity in the past, biological transformation ratio is 30-60% only, have in addition have only about 10%, thereby cause and yield poorly the cost height.
The fine-variety breeding and the high yield cultivating method that the purpose of this invention is to provide a kind of Cordyceps militaris (L.) Link..It guarantees that effectively the bacterial classification of seed selection is pure, is difficult for degenerating, and obviously improves biological transformation ratio, shortens the production cycle, significantly reduces production costs.
The object of the present invention is achieved like this: the original strain that the Cordyceps militaris (L.) Link. that will newly gather obtains by conventional monospore or many spores separation method, and through artificially breeding and high-yield culturing.The screening of its seed selection process three phases: female kind of stage: original strain is seeded on the test tube or the solid medium in the culture dish of sterilization, lucifuge was cultivated 7-10 days, be orange after seeing light, it is long to select mycelia fine hair according to colonial morphology and microscopy, uniformity, the mycelia branch many and grow, in obtuse angle, the rounded bacterial strain of conidium gives over to female the kind and carries out the first time and screen; The original seed stage: with above-mentioned female the kind on the liquid nutrient medium that is seeded in the triangular flask of sterilization after vibration, lucifuge are cultivated 5 days,, be orange after seeing light, select bacterial strain by above-mentioned standard and give over to original seed, carry out programmed screening again through static cultivation in 2 days; The cultivar stage: above-mentioned original seed is seeded on the liquid nutrient medium in the Cans of sterilization, cultivates, see after light 1-2 days to be orange, select bacterial strain by above-mentioned standard and give over to cultivar, screen for the third time through 10-12 days lucifuges.Spawn rejuvenation: the bacterial classification through three screenings carries out 3-5 repetition experiment in cultivation again, selects the female kind of cultivation at last, then substratum is increased pupa alive or larva body or the milk powder of 1-5%, carries out rejuvenation.The high-yield culturing of Cordyceps militaris (L.) Link.: 2 milliliters of above-mentioned cultivars of inoculation on the solid medium of each culturing bottle in sterilisable chamber through rejuvenation, lucifuge was cultivated 10-20 days, cover with mycelia and move into cultivating chamber, indoor relative humidity 85-90%, CO2 0.03-0.05%, illumination 2,000 5-2 ten thousand Lux can grow sporophore through 3-5 days, did not distribute in time in a large number at thecaspore and gathered.Gather behind the first stubble entity, media surface is cleaned out with aseptic cutter, expose white hypha, prick the hole thereon or draw 0.5 centimetre of dark ditch, inject 10-20 milliliter nutritive medium, bottleneck is sealed, under above-mentioned similarity condition, can be grown the second stubble entity, can grow the 3rd, the 4th stubble entity more continuously with quadrat method and condition through cultivation in 7-10 days.Used culture medium prescription is as follows in each stage of seed selection and high yield cultivating method: the female kind stage culture medium prescription of seed selection: potato 21-25%, glucose or sucrose 2.1-2.5%, agar 2.1-2.5%, peptone 0.2-0.25%, potassium primary phosphate 0.15-0.2%, dipotassium hydrogen phosphate 0.11-0.15%, sal epsom 0.1-0.15%, VBl 0.001-0.002%, live silkworm chrysalis or larva 1-5%, with soil extract to 100%; Original seed stage culture medium prescription is: potato 21-25%, glucose or sucrose 2.1-2.5%, potassium primary phosphate 0.15-0.2%, dipotassium hydrogen phosphate 0.11-0.15%, sal epsom 0.1-0.15%, VBl 0.001-0.002%, silkworm chrysalis alive or larva 1-5%, with soil extract to 100%, its pH value is 8-8.5; Cultivar stage culture medium prescription is: potato 21-25%, glucose or sucrose 2.1-2.5%, potassium primary phosphate 0.15-0.2%, dipotassium hydrogen phosphate 0.11-0.15%, sal epsom 0.1-0.15%, VBl 0.001-0.002%, milk powder 2-3%, with soil extract to 100%, its pH value is 8-8.5; The used solid culture based formulas of high-yield culturing is: rice 30-50 gram adds 1.7 times of murphy juices, transfers PH8-8.5; Rice 30-50 gram adds 1.6 times in water again with milk powder 2-5 gram, transfers pH value 8-8.5; Rice 30-50 gram adds pupa albumen 2-5 gram, adds 1.6 times in water again, transfers PH8-8.5.Institute's Ensure Liquid liquid formula is: potato 21-25%, glucose or sucrose 2.1-2.5%, potassium primary phosphate 0.15-0.2%, dipotassium hydrogen phosphate 0.11-0.15%, sal epsom 0.1-0.15%, VBl 0.001-0.002%, silkworm chrysalis alive or larva 1-5%, with soil extract to 100%, its pH value is 8-8.5.
Because the present invention is after existing Cordyceps militaris (L.) Link. bacterium and artificial culture sporophore method thereof are carried out a large amount of experimental studies, the imperfect stage of confirming Cordyceps militaris (L.) Link. belongs to Cephalosporium (ce-phalosporlum mitilaris kob), so it provides reliable foundation for Cordyceps militaris spawn seed selection, purification, rejuvenation.Under study for action, found that also there be " heterokaryosis " in Cordyceps militaris (L.) Link., this heterokaryosis has a direct impact the generation of sporophore, through for a long time a large amount of cultivation experiments, sum up fine-variety breeding and the high yield cultivating method of above-mentioned Cordyceps militaris (L.) Link., obtained result in unexpected positive.This method is by behind female kind, the screening and rejuvenation of original seed, cultivar three phases, obtain kind of the metastable pure bacterial classification of property, be difficult for degenerating, this bacterial classification is after obtaining the first stubble entity, append nutritive medium again at its solid medium, can obtain continuously second and third, four stubble entities, biological transformation ratio is reached more than 150%, shorten the production cycle more than 20 days, reduce production costs about 40%.
The invention will be further described below in conjunction with specific implementation method.
Implementation method one
The seed selection of excellent species:
Female kind stage: the original strain that obtains of spore separation technology is seeded on the solid medium in the test tube (or culture dish) of sterilization routinely, after lucifuge under the 20-25 ℃ of temperature is cultivated 7-10 days, see and be orange in light 1-2 days, it is long to select mycelia fine hair according to colonial morphology and microscopy, uniformity, and the mycelia branch is many and grow, in obtuse angle, the rounded bacterial strain of conidium gives over to female the kind, carries out the screening first time, and all the other are eliminated.Its solid culture based formulas is: potato 210 grams, sucrose 25 grams, agar 22 grams, peptone 2 grams, potassium primary phosphate 1.8 grams, dipotassium hydrogen phosphate 1.2 grams, sal epsom 1 gram, 10 milligrams of VBl, silkworm chrysalis 10 grams of living, and with soil extract to 1000 milliliter.The original seed stage: with above-mentioned female the kind on the liquid nutrient medium that is seeded in the triangular flask of sterilization, under 20-25 ℃ of condition, after the vibration lucifuge is cultivated 5 days, again through static cultivation in 2 days, be orange after seeing light, select bacterial strain by above-mentioned standard and give over to original seed, all the other are eliminated.Its culture medium prescription: potato 220 grams, sucrose 24 grams, potassium primary phosphate 2 grams, dipotassium hydrogen phosphate 1 gram, sal epsom 1.5 grams, 15 milligrams of VBl, silkworm larva 50 grams of living, with soil extract to 1000 milliliter, transfer PH8-8.5.The cultivar stage: above-mentioned original seed is seeded on the liquid nutrient medium of the Cans through sterilizing, cultivates through 10-12 days lucifuges under 20-25 ℃ of temperature, see after light 1-2 days to be orange, select bacterial strain by above-mentioned standard and give over to cultivar, all the other are eliminated.Its culture medium prescription is: potato 250 grams, sucrose 20 grams, potassium primary phosphate 2 grams, dipotassium hydrogen phosphate 1 gram, sal epsom 1 gram, 20 milligrams of VBl, milk powder 30 grams, with soil extract to 1000 milliliter, transfer PH8-8.5, the bacterial classification that obtains through three screenings carries out 3 repetition experiments in cultivation again, select the female kind of cultivation at last, pupa alive or the larva body with its substratum increase by 1% carries out rejuvenation then.High-yield culturing: in sterilisable chamber, 2 milliliters of above-mentioned cultivars through rejuvenation are seeded on the solid medium of each culturing bottle, under 24 ± 2 ℃ of temperature, relative air humidity 60-65%, CO2 concentration 0.05-0.1% lucifuge was cultivated 10-20 days, move into cultivating chamber after covering with mycelia, temperature is at 21 ± 2 ℃, relative air humidity 85-90%, CO2 concentration 0.03-0.05%, bright uniform scattered light irradiation is arranged, illumination 2,000 5-2 ten thousand Lux, ventilate 3-4 every day, each about 20 minutes, the dazzling sporophore deformity that prevents can grow sporophore through 3-5 days on bottle cap, the first stubble entity of in time gathering when thecaspore does not distribute in a large number.After the first stubble entity is gathered, its substratum taking-up is cleaned out media surface with aseptic cutter, expose white hypha, put back to then in the culture bottle, how many dazzling or stroke 0.5 centimetre of dark ditches adds 10-20 milliliter nutritive medium according to its water content on its surface, bottleneck is sealed, be placed under 24 ± 2 ℃ of temperature relative air humidity 60-65%, CO
2Concentration 0.05-0.1%, lucifuge was cultivated 5-7 days, after covering with mycelia, move into cultivating chamber, temperature is at 21 ± 2 ℃, relative humidity 85-90%, CO2 concentration 0.03-0.05%, illumination 2,000 5-2 ten thousand Lux can grow sporophore through 3-5 days, the second stubble entity of in time gathering when thecaspore does not distribute in a large number is according to the 3rd, the 4th stubble entity of can gathering continuously with quadrat method and condition.The culture medium prescription of the first stubble entity is: rice 30 grams, add 1.7 times murphy juice, PH8-8.5, institute's Ensure Liquid liquid formula is on the solid medium of 2-4 stubble: potato 250 grams, sucrose 20 grams, potassium primary phosphate 1.6 grams, dipotassium hydrogen phosphate 1.4 grams, sal epsom 1 gram, 20 milligrams of VBl, silkworm chrysalis 30 grams of living, with soil extract to 1000 milliliter, PH8-8.5.
Implementation method two
Cordyceps militaris excellent species seed selection: adopt as method steps and identical breeding standard that implementation method one is identical,, obtain to plant the metastable cultivar of property through rejuvenation again through female kind of stage, original seed stage, cultivar stage.The culture medium prescription in each stage is: female kind of stage: potato 250 grams, sucrose 20 grams, agar 25 grams, peptone 2.5 grams, potassium primary phosphate 2 grams, dipotassium hydrogen phosphate 1 gram, sal epsom 1.5 grams, 20 milligrams of VBl, live silkworm chrysalis or larva 50 grams, with soil extract to 1000 milliliter; The original seed stage: potato 240 grams, sucrose 25 grams, potassium primary phosphate 1.8 grams, dipotassium hydrogen phosphate 1.2 grams, sal epsom 1.5 grams, 15 milligrams of VBl, silkworm chrysalis 30 grams of living, with soil extract to 1000 milliliter; The cultivar stage: potato 230 grams, glucose 20 grams, potassium primary phosphate 1.6 grams, dipotassium hydrogen phosphate 1.4 grams, sal epsom 1.2 grams, 10 milligrams of VBl, milk powder 30 grams, with soil extract to 1000 milliliter, PH8-8.5, the cultivation chosen is female plants, and its substratum is added 5% milk powder and carries out rejuvenation.High yield cultivating method step condition is with implementation method one, its first stubble entity culture medium prescription is: rice 50 grams, the milk powder (or pupa albumen) that adds the 2-5 gram, add 1.6 times water again, transfer PH8-8.5,2-4 stubble substratum institute Ensure Liquid liquid formula is: potato 220 grams, glucose 25 grams, potassium primary phosphate 2 grams, dipotassium hydrogen phosphate 1 gram, sal epsom 1.5 grams, 10 milligrams of VBl, live silkworm chrysalis or larva 50 grams, and with soil extract to 1000 milliliter, PH8-8.5.
Claims (8)
1, a kind of seed selection of cordyceps militaris excellent species and high yield cultivating method, Cordyceps militaris (L.) Link. (cordycepsmilitaris (L) link) has another name called Cordyceps militaris (L.) Link., this bacterial classification has been deposited in Beijing China Committee for Culture Collection of Microorganisms common micro-organisms center on April 1st, 1993, preserving number CGMCC NO0193, comprise that the Cordyceps militaris (L.) Link. that will newly gather passes through the original strain of conventional monospore or the acquisition of many spores separation method, carry out artificially breeding and high-yield culturing, its operation steps is as follows:
1.1, the seed selection of cordyceps militaris excellent species: with original strain through female kind of stage, original seed stage and three screenings of cultivar stage after, carry out rejuvenation;
1.1.1, female kind of stage: earlier original strain is seeded on the test tube or the solid medium in the culture dish of sterilization, lucifuge was cultivated 7-10 days, be orange after seeing light, it is long to select mycelia fine hair according to colonial morphology and microscopy, uniformity, the mycelia branch many and grow, in obtuse angle, the rounded bacterial strain of conidium gives over to female the kind and carries out the first time and screen;
1.1.2, the original seed stage: with above-mentioned female the kind on the liquid nutrient medium that is seeded in the triangular flask of sterilization after vibration, lucifuge are cultivated 5 days,, be orange after seeing light, select bacterial strain by above-mentioned standard and give over to original seed, carry out programmed screening again through static cultivation in 2 days;
1.1.3, the cultivar stage: above-mentioned original seed is seeded on the liquid nutrient medium in the Cans of sterilization, cultivates, see after light 1-2 days to be orange, select bacterial strain by above-mentioned standard and give over to cultivar, screen for the third time through 10-12 days lucifuges;
1.1.4, spawn rejuvenation: the bacterial classification through three screenings carries out repeating experiment in cultivation for 3-5 time again, selects the female kind of cultivation at last, then substratum is increased pupa alive or larva body or the milk powder of 1-5%, carries out rejuvenation;
1.2, the high-yield culturing of Cordyceps militaris (L.) Link.;
1.2.1, in sterilisable chamber 2 milliliters of above-mentioned cultivars of inoculation on the solid medium of each culturing bottle through rejuvenation, lucifuge was cultivated 10-20 days, covered with mycelia and moved into cultivating chamber, indoor relative humidity 85-90%, CO
20.03-0.05%, illumination 2,000 5-2 ten thousand lux can grow sporophore through 3-5 days, did not distribute in time in a large number at thecaspore and gathered;
1.2.2, behind the first stubble entity of gathering, media surface is cleaned out with aseptic cutter, expose white hypha, prick the hole thereon or draw 0.5 centimetre of dark ditch, inject 10-20 milliliter nutritive medium, bottleneck is sealed, under above-mentioned similarity condition, can be grown the second stubble entity, can grow the 3rd, the 4th stubble entity more continuously with quadrat method and condition through cultivation in 7-10 days.
2, method according to claim 1, it is characterized in that the female kind stage culture medium prescription of seed selection: potato 21-25%, glucose or sucrose 2.1-2.5%, agar 2.1-2.5%, peptone 0.2-0.25%, potassium primary phosphate 0.15-0.2%, dipotassium hydrogen phosphate 0.11-0.15%, sal epsom 0.1-0.15%, VBl 0.001-0.002%, live silkworm chrysalis or larva 1-5%, with soil extract to 100%.
3, method according to claim 1, it is characterized in that seed selection original seed stage culture medium prescription is: potato 21-25%, glucose or sucrose 2.1-2.5%, potassium primary phosphate 0.15-0.2%, dipotassium hydrogen phosphate 0.11-0.15%, sal epsom 0.1-0.15%, VBl 0.001-0.002%, silkworm chrysalis alive or larva 1-5%, with soil extract to 100%, its pH value is 8-8.5.
4, method according to claim 1, it is characterized in that breeding and cultivating kind stage culture medium prescription is: potato 21-25%, glucose or sucrose 2.1-2.5%, potassium primary phosphate 0.15-0.2%, dipotassium hydrogen phosphate 0.11-0.15%, sal epsom 0.1-0.15%, VBl 0.001-0.002%, milk powder 2-3%, with soil extract to 100%, its pH value is 8-8.5.
5, method according to claim 1 is characterized in that the used solid culture based formulas of high-yield culturing is: rice 30-50 gram adds 1.7 times of murphy juices, transfers PH8-8.5.
6, method according to claim 1 is characterized in that the used solid culture based formulas of high-yield culturing is: rice 30-50 gram adds 1.6 times in water again with milk powder 2-5 gram, transfers pH value 8-8.5.
7, method according to claim 1 is characterized in that the used solid culture based formulas of high-yield culturing is: rice 30-50 gram, add pupa albumen 2-5 gram, and add 1.6 times in water again, transfer PH8-8.5.
8, method according to claim 1, it is characterized in that the prescription that high-yield culturing appends nutritive medium is: potato 21-25%, glucose or sucrose 2.1-2.5%, potassium primary phosphate 0.15-0.2%, dipotassium hydrogen phosphate 0.11-0.15%, sal epsom 0.1-0.15%, VBl 0.001-0.002%, silkworm chrysalis or larva 1-5% live, with soil extract to 100%, its pH value is 8-8.5.
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